Purification of porphobilinogen deaminase from Euglena gracilis and studies of its kinetics.
1. Porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] from Euglena gracilis was purified more than 200-fold. 2. The enzyme has a molecular weight of 41 000 +/- 2000, does not contain a chromophoric prosthetic group, and appears not to require metal ions for activity. 3. The stoicheiometry of the overall reaction at pH 7.4 was shown to be: 4 Porphobilinogen leads to uroporphyrinogen-I + 4 NH4+. This stoicheiometry for porphobilinogen and uroporphyrinogen was also observed over a wide range of pH values. 4. Initial-velocity studies showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. 5. Vmax. varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 6.1 and 8.9 are important for catalysis. A plot of Vmax./Km against pH showed a single ionization (pK 8.2) to influence binding of substrate.
D000642:Ammonia-Lyases / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D005056:Euglena gracilis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D011163:Hydroxymethylbilane Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011162:Porphobilinogen / Q000378:metabolism; D013696:Temperature
D011162:Porphobilinogen; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases